The differential distribution of hyaluronic acid in the layers of human atheromatic aortas is associated with vascular smooth muscle cell proliferation and migration
Introduction
Atherogenesis, the complex cascade of events leading to the formation of atheromatic plaques, depends on the interaction between several cell types, growth factors, cytokines and molecules of the extracellular matrix [1]. Among different cell types, vascular smooth muscle cells (VSMC) play a major role in the formation of atherosclerotic lesions 2, 3. In the normal human arteries, susceptible to atherosclerosis, VSMC reside mainly in the tunica media in a quiescent state and express a variety of differentiation-specific genes important to maintain the physiological regulation of vessel tone and blood pressure [4]. During the inflammatory injury seen in the early stages of atherosclerosis, VSMC migrate from the tunica media to the tunica intima. These migrating VSMC exhibit a synthetic phenotype with subcellular organization designed to support rapid cell growth and proliferation. Among several growth factors and cytokines platelet-derived growth factor (PDGF) has been shown to be essentially involved in the progression of atherosclerosis [1]. Extensive in vitro studies revealed that PDGF-BB stimulates the proliferation of VSMC, endothelial cells and fibroblasts 1, 5, 6. However, recent data suggest that this growth factor is only a weak mitogen for VSMC in vivo 7, 8, 9but it induces their migration from the tunica media into the tunica intima [1].
Among different molecules involved in atherogenesis the glycosaminoglycans (GAGs) have been reported to contribute in some key events leading to the formation of atherosclerotic lesions [10]. Extracellular matrix GAGs provide structural links between fibrous and cellular elements, contribute to viscoelastic properties, regulate permeability and retention of plasma components within the matrix 10, 11, inhibit vascular cell growth [12], affect hemostasis and platelet aggregation [13]and interact with lipoproteins [14]. Specific GAGs such as dermatan sulfate and chondroitin sulfates A and C have been shown to increase in relation to early fibrous plaque formation in the tunica media [15], the coronary arteries of humans [16], or in the coronary arteries of rhesus monkeys [17]. In the tunica media of atherosclerotic human aortas, an increase in dermatan sulphate and chondroitin sulphate has been reported [18]but others did not observe any changes [19].
Hyaluronic acid (HA), a mainlike GAG, appears to be of particular interest, since tissues enriched in HA may undergo expansion due to the ability of the molecule to bind large amounts of water, thus creating a loose, hydrated micro environment that facilitates cell migration and proliferation, two critical events in atherogenesis [1]and arterial development [20]. In this context, it has been reported that HA is secreted by arterial smooth muscle cells [21], HA-synthesizing enzymes increase during arterial smooth muscle cell proliferation [22]and HA decreases with atherosclerotic involvement of the tunica media 15, 16, 17, 23.
It was recently shown that PDGF-BB specifically stimulates VSMC, in culture, to secrete a 340-kDa acidic glycan molecule (HA-340), 82% of which exhibits structural homology with HA and inhibits VSMC proliferation, in vitro [24]. Here, evidence is presented that the HA which is expressed in all layers of the human aorta and the atherosclerotic plaque has the same molecular mass as that identified in VSMC, in culture. Furthermore, we show that the human aorta HA affects the PDGF-induced VSMC proliferation and migration through an artificial basement membrane (BM), in vitro. The abundance of this molecule in the tunica media as compared with the low levels in the tunica intima and atherosclerotic plaque correlates with its function as a negative control element of VSMC proliferation and as a positive control element of VSMC migration during the development of atherosclerotic lesions.
Section snippets
Isolation and purification of glycans
Biopsies of human aortas (upper thoracic level and its adjacent part of the aortic arch), from male adults (n=4, age 30–35 years) were obtained at autopsy within 6 h of death by accident and were kindly provided by the Department of Forensic Medicine, School of Medicine, Aristotle University of Thessaloniki, Greece. The medical history of the individuals was free from diseases, such as hypertension and diabetes, but autopsy revealed that there was significant plaque development in their aorta.
Isolation and purification of the total glycans
Human atheromatic aortas, obtained at autopsy, were dissected in the atheromatic plaque, the tunica intima, the tunica media and the tunica adventitia. The atherosclerotic lesions were identified as fibrofatty. Total glycans were isolated after homogenization of tissue samples, delipidation and sequential treatment with pronase, DNase and alkali borohydride. Purification of the glycans from the digestion products was achieved on a Sephadex G-25 column as a single peak, with 90% recovery (data
Discussion
GAGs are a group of complex macromolecules that are expressed in a wide range of tissues and compose an essential part of the extracellular matrix. They also play a pivotal role in sequestering and presenting a wide range of growth factors and cytokines to a diverse range of responding cell types [36]. We have recently reported that PDGF specifically stimulates proliferating VSMC to secrete HA-340, a 340-kDa HA-like molecule [24]. This increased production of HA-340 was restricted to
Acknowledgements
E. Papakonstantinou was supported by a fellowship from the European Commission (ERBFMBI-CT-96-0871).
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